B. Protein Blotting

A general protocol for sample preparation.

Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

(Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.

Wash membrane again four times for 5 min each in TBST.

The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

Specificity / Sensitivity

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence of human GP130. Antibodies are purified by protein A and peptide affinity chromatography.

Background

GP130 is a signal-transducing subunit shared by the receptors for the IL-6 family of cytokines (1-3). The binding of a ligand to its receptor induces the dimerization of GP130, leading to activation of the Jak tyrosine kinase and to tyrosine phosphorylation of GP130. These events lead to the activation of multiple signal-transduction pathways, such as the Stat, Ras-MAPK and PI3 kinase pathways, whose activation is controlled by distinct regions of GP130 (4-7).